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This lecture covers the fundamentals of protein separation, including protein charge dependence on pH, isoelectric focusing, SDS Polyacrylamide Gel Electrophoresis, capillary electrophoresis, and protein identification by mass spectrometry. Various techniques such as continuous flow IEF chip, off-gel IEF, and free flow electrophoresis are discussed, along with the resolution in capillary electrophoresis and 2D PAGE for protein expression profiling.